Identification and characterization of α1 -antitrypsin in fibrin clots.

BACKGROUND AND OBJECTIVES: Preliminary studies indicated that α1 -antitrypsin (A1AT) is the most abundant protein that is non-covalently bound to fibrin clots prepared from plasma. The aim of this study was to identify and characterize fibrin(ogen)-bound A1AT. METHODS AND RESULTS: Plasma clots were prepared and extensively washed with saline. Clot-bound A1AT could only be extracted using denaturing agents such as urea, thiourea or SDS, pointing to an apparently strong association. Purified fibrinogen, but still containing A1AT as a contaminant, was gel filtered, which showed that the A1AT was bound to fibrinogen. A specific ELISA detected the presence of A1AT-fibrinogen complexes in both purified fibrinogen and pooled normal plasma. Finally, fibrin(ogen)-Sepharose chromatography indicated that A1AT purified from plasma contained a small fraction of fibrin(ogen)-binding A1AT. To study the inhibitory activity of fibrin(ogen)-bound A1AT, both fibrinogen containing A1AT and washed plasma clots were incubated with increasing amounts of elastase. SDS-PAGE and Western blotting showed under both conditions the generation of the A1AT-elastase complex as well as cleaved A1AT. The inhibitory activity of fibrin(ogen)-bound A1AT was also demonstrated by measuring elastase-induced lysis of fibrin clots. CONCLUSION: Fibrin clots contain strongly bound A1AT, which is functionally active as a serine protease inhibitor (serpin). This A1AT might play a role in the local regulation of proteases involved in coagulation or fibrinolysis and represent a novel link between the inflammatory and hemostatic systems.

Binding of carboxypeptidase N to fibrinogen and fibrin.

The ultimate step in the blood coagulation cascade is the formation of fibrin. Several proteins are known to bind to fibrin and may thereby change clot properties or clot function. Our previous studies identified carboxypeptidase N (CPN) as a novel plasma clot component. CPN cleaves C-terminal lysine and arginine residues from several proteins. The activity of CPN is increased upon its proteolysis by several proteases. The aim of this study is to investigate the presence of CPN in a plasma clot in more detail. Plasma clots were formed by adding thrombin, CaCl(2) and aprotinin to citrated plasma. Unbound proteins were washed away and non-covalently bound proteins were extracted and analyzed with 2D gel electrophoresis and mass spectrometry. The identification of CPN as a fibrin clot-bound protein was verified using Western blotting. Clot-bound CPN consisted of the same molecular forms as CPN in plasma and its content was approximately 30 ng/ml plasma clot. Using surface plasmon resonance we showed that CPN can bind to fibrinogen as well as to fibrin. In conclusion, CPN binds to fibrinogen and is present in a fibrin clot prepared from plasma. Because CPN binds to a fibrin clot, there could be a possible role for CPN as a fibrinolysis inhibitor.

Evidence for an enhanced fibrinolytic capacity in cirrhosis as measured with two different global fibrinolysis tests.

BACKGROUND AND OBJECTIVES: It has been known for a long time that cirrhosis is associated with hyperfibrinolysis, which might contribute to an increased risk and severity of bleeding. However, recent papers have questioned the presence of a hyperfibrinolytic state in cirrhotic patients and postulated a rebalanced system owing to concomitant changes in both pro- and anti-fibrinolytic factors. Therefore we re-investigated the fibrinolytic state of cirrhotic patients using two different overall tests including a recently developed test for global fibrinolytic capacity (GFC) using whole blood. PATIENTS AND METHODS: Blood was collected from 30 healthy controls and 75 patients with cirrhosis of varying severity (34 Child-Pugh A, 28 Child-Pugh B and 13 Child-Pugh C). The plasma clot lysis time (CLT), which is inversely correlated with fibrinolysis, was determined as well as the GFC. RESULTS: The mean CLT was 74.5 min in the controls and decreased significantly to 66.9 min in Child-Pugh class A patients, 59.3 min in class B patients and 61.0 min in class C patients, and hyperfibrinolysis existed in 40% of the patients. The median GFC was 1.7 µg mL(-1) in the controls and increased significantly to 4.0 µg mL(-1) in Child-Pugh class A patients, 11.1 µg mL(-1) in class B patients and 22.5 µg mL(-1) in class C patients, and hyperfibrinolysis existed in 43% of the patients. Taken together, 60% of the patients showed hyperfibrinolysis in at least one of the two global assays. CONCLUSION: A rebalanced fibrinolytic system may occur, but hyperfibrinolysis is found in the majority of patients with cirrhosis.